Methods for determining ldl cholesterol treatment

ABSTRACT

Disclosed is a personalized diagnostic and treatment solution for cardiovascular disease. The invention comprises an extended CVD risk assessment panel, combining tests for traditional and new important risk markers, and methods for devising a personalized treatment plan for a patient via the use of a CVD diagnosis and treatment protocol algorithm. The new important risk markers include HDL subpopulation profile by two-dimensional gel-electrophoresis, plasma sterols, direct measurement of sdLDL-C, determination of CRP molecular forms, glycated albumin as a percentile of total albumin, and other specialized testing pertaining to apolipoprotein E and Factor V Leiden genotyping, NT-proBNP and adiponectin. This solution provides a more complete risk assessment of an individual than merely measuring traditional CVD risk markers, and enables the healthcare practitioner to optimize therapy for patients with or without established CVD. This solution presents the advantages of greater accuracy, savings in time and cost over existing testing and treatment methods.

REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. non-provisionalapplication Ser. No. 13/945,436, filed Jul. 18, 2013, which is acontinuation of U.S. non-provisional application Ser. No. 13/658,387,filed Oct. 23, 2012, which is a continuation of U.S. non-provisionalapplication Ser. No. 12/472,351, filed on May 26, 2009, which claims thebenefit of and priority to U.S. provisional patent applications havingSer. No. 61/056,163, filed on May 27, 2008, and 61/084,909, filed onJul. 30, 2008. Each of the above-referenced applications is incorporatedby reference.

TECHNICAL FIELD

The present invention relates generally to cardiovascular disease (CVD)risk assessment, diagnosis and treatment thereof. Specifically, thepresent invention pertains to a diagnosis and personalized treatmentsolution based on results from testing utilizing an extended CVD riskassay panel that measures the combination of traditional risk factorsand new important risk markers, and analysis of said results via a CVDdiagnosis and treatment protocol algorithm in order to assess CVD risk,evaluate efficacy of drug therapy, and optimize therapy.

BACKGROUND

Cardiovascular disease, which includes coronary heart disease (CHD) andstroke, is the leading cause of death and disability in developedcountries of the world. CVD is caused by clogging of arteries. Majoraccepted risk factors for CVD include age, gender, hypertension,smoking, diabetes, elevated blood low density lipoprotein cholesterol(LDL-C), and decreased blood high density lipoprotein cholesterol(HDL-C).

In order to assess CVD risk, an assay panel is utilized for testing anindividual's risk factors. A typical risk assessment screening testincludes measuring fasting levels of total cholesterol (TC),triglycerides (TG), HDL-C, calculated LDL-C, hemoglobin Alc, andglucose. However, such a risk assessment panel is limited to traditionalrisk factors and does not provide a complete assessment of CVD risk, orways to optimize treatment.

There are other tests for general metabolic factors of kidney, liver,muscle and thyroid function that are often not performed. These includeblood urea nitrogen or BUN, creatinine, BUN/creatinine ratio, albumin,globulin, albumin/globulin ratio, alkaline phosphatase, liver enzymesAST and ALT, creatine kinase (CK), thyroid stimulating hormone (TSH),glomerular filtration rate (GFR), calcium, total protein, totalbilirubin, sodium, potassium, chloride, carbon dioxide and uric acid;however, these tests are not always consistently used, despite theirimportance in ruling out secondary causes of lipid abnormalities.

There are other tests for heart disease risk, such as testing for levelsof non-HDL cholesterol, and very low density lipoprotein cholesterol(VLDL-C), and the total cholesterol/HDL cholesterol ratio; however,these test are not consistently used in CVD risk assessment.

In addition there are specialized tests for CVD risk which includetesting for levels of direct LDL cholesterol, small dense LDLcholesterol (sdLDL-C), lipoprotein (a) or Lp(a), apolipoprotein A-I(apoA-I), apolipoprotein B (apoB), fibrinogen, and homocysteine.

There are specialized testing for C-reactive protein (CRP with a highlysensitive test or hsCRP), lipoprotein associated phospholipase A2(LpPLA2), N-terminal pro-brain natriuretic peptide (NT-proBNP), insulin,adiponectin, and glycosylated hemoglobin (HbA1C); however, they are notwidely or consistently used in CVD risk assessment.

There are tests for plasma sterols, such as lathosterol, desmosterol,beta sitosterol, campesterol, and cholestanol; however, they are notwidely or consistently used in CVD risk assessment.

In addition, genotyping of apolipoprotein E and Factor V Leiden providesvaluable information about CVD and dementia risk, as well as risk ofclot formation, but it is generally not utilized in CVD risk assessment.

Lipoproteins in serum or plasma are complexes of various lipids andproteins. The major lipoproteins based on ultracentrifugal separationare chylomicrons (CM), very low density lipoproteins (VLDL), low densitylipoproteins (LDL), and high density lipoproteins (HDL). Theselipoproteins can also be fractionated by size, protein components,electrophoretic mobility, or any combination of these. If plasma issubjected to two separation methods, the major lipoprotein classes canfurther be separated into subclasses. These subclasses differ from eachother in size, charge, chemical composition, and patho-physiologicimportance.

The ultracentrifugal (UC) method separates lipoproteins based on theirrespective specific flotation rate (density) into HDL, LDL, VLDL, andCM, in decreasing density, respectively. However, the UC method is verylabor intensive, requires a specialized laboratory, and is veryexpensive. Moreover, the high separating force (100,000 times of normalgravity) used in this method affects the integrity of the lipoproteinparticles, therefore, the UC method produces a significant amount ofartifacts (in vitro altered lipoproteins) that affect the result. Inaddition, even finer fractionation of the sample is necessary forrelating fractions to diseases, but the additional fractionation stepincreases the production of artifacts. For these reasons, separation ofplasma or serum lipoproteins by ultracentrifugation is neither feasiblenor ideal for clinical diagnostic evaluation of plasma lipoproteins andcardiovascular disease (CVD) risk.

Size exclusion separation of HDL, as with fast low pressure liquidchromatography (FPLC), has no adequate resolution, needs a largequantity of plasma, and produces artifacts due to the excessive dilutionof the plasma. Magnetic nuclear resonance (NMR) is anothercharacterization technique that is widely used, primarily because of itsspeed; however, it is unclear how to interpret NMR signal data or whatthese data represent. One-dimensional non-denaturing gel electrophoresisis also used for characterizing lipoproteins, such as HDL and LDL. Withthis method, lipoproteins are separated only by size. With this method,separation between the pre13-mobility and a-mobility HDL particles isnot achievable. Thus, this method does not allow for the accurateassessment of a-1 HDL, pre13-2, or pre13-1 HDL, all of which areimportant particles for CVD risk assessment.

HDL can protect against atherosclerosis in several ways. The most citedHDL function to protect against atherosclerosis is its participation inreverse cholesterol transport. During this process, HDL removescholesterol from macrophages in the vessel wall, preventing thetransformation of macrophages into foam cells, eventually preventing thebuild-up of fatty streaks and plaque in the vessel wall. The cholesterolthat originated in the macrophages is then carried by HDL to the liverfor ultimate excretion into the bile.

HDL is also an anti-oxidant and anti-inflammatory agent. Oxidativestress can cause inflammation in the vessel wall. The protein and lipidcomponents of HDL can prevent LDL oxidation. This is a very importantfunction because oxidized LDL is the major carrier of cholesterol tomacrophages present in the vessel wall. Moreover, HDL hasanti-inflammatory functions and participates in the immune response.

The different HDL particles have different pathophysiological relevance.The many different functions of HDL are not distributed evenly among thevarious HDL subclasses. The best illustration of this is that cells haveseveral ways to remove excess cholesterol. The different HDL particlesspecifically interact with the different pathways depending on celltype, the expressed receptor protein type on the surface of the cell,and cellular cholesterol content. Also, the different HDL particlesparticipate differently in the anti-oxidation and anti-inflammationprocesses based on the lipid and protein composition of the HDLparticles.

Data from HDL- and CVD-related population-based studies reveal thefollowing:

-   -   For every 1 mg increase in HDL cholesterol, there is a 2-3%        reduction in CVD risk. [“High-density Lipoprotein Cholesterol        and Cardiovascular Disease. Four Prospective American Studies.”,        Gordon, D. J. et. al., Circulation, 1989, January; 79(1):8-15].    -   In the Framingham Offspring Study, in men free of CHD (n=1277)        and men with CHD (n=169), for every 1 mg/dl increase in a-1 HDL        there was a 26% decrease in risk of CHD (probability        (“p”)<0.001), and HDL particles were superior to HDL-C values in        predicting prevalence of CHD. [“High-density Lipoprotein        Subpopulation Profile and Coronary Heart Disease Prevalence in        Male Participants of the Framingham Offspring Study”,        Asztalos, B. F., et. al., Arterioscler Thromb Vasc Biol. 2004,        November; 24(11):2181-7. Epub 2004 Sep. 23].    -   Patients with CHD have lower HDL-C due to decreases in the large        cholesterol-rich a-1 HDL (−39%) and increases in the small        lipid-poor alpha a-3 HDL (+29%) and pre-B1 HDL (+16%) as        compared to age- and gender-matched controls. [“Distribution of        ApoA-I-containing HDL Subpopulations in Patients with Coronary        Heart Disease”, Asztalos, B. F., et. al., Arterioscler Thromb        Vasc Biol., 2000, December; 20(12):2670-6; and “High-density        Lipoprotein Subpopulation Profile and Coronary Heart Disease        Prevalence in Male participants of the Framingham Offspring        Study”, as cited above].    -   In the Veterans Affairs HDL Intervention Study (VA-HIT), low        levels of a-1 and a-2 HDL predicted recurrent CHD events (n=398)        versus no recurrence (n=1097) in men selected for low HDL C        (less than 40 mg/dl and presence of CHD. Low a-1 HDL was the        most significant parameter predicting recurrence (p<0.001).        [“Value of High-Density Lipoprotein (HDL) Subpopulations in        Predicting Recurrent Cardiovascular Events in the Veterans        Affairs HDL Intervention Trial”, Asztalos, B. F., et. al.,        Arterioscler Thromb Vasc Biol., 2005, October;        25(10):2185-2191].

Two-dimensional gel electropheresis is a separation method, based on thecombination of two principles of electrophoretic separation (in thefirst dimension, particles are separated by charge and in the seconddimension by size) that is very useful for reproducibly separating HDLparticles with high resolution. The method is quantitative byutilization of protein immuno-localization and image-analysis. As aresult of employing this two-dimensional HDL separation method,different HDL particles have been associated with CVD risk inpopulation-based cross-sectional studies. The two-dimensional gelelectrophoresis technology is also useful in the diagnosis of thehomozygous and heterozygous state for rare inherited HDL disorders, suchas apoA-I/C-IIIA-IV, apoA-I/C-III deficiency, isolated apoA-Ideficiency, ABCA1 deficiency, LCAT deficiency, SRB1 deficiency, CETPdeficiency, lipoprotein lipase deficiency, hepatic lipase deficiency,and endothelial lipase deficiency. Based on the scans generated usingthis technique, it has become possible to differentiate among thevarious HDL particles; this allows for very precise evaluation of theseverity of CVD-risk. Patients who are carriers of one normal and onedamaged gene (referred to as heterozygotes) of the above list also havereduced levels of HDL and premature CVD. Patients who are carriers oftwo damaged genes (referred to as homozygotes) of the above listgenerally have a very high risk for premature CHD. Patients with ABCA1mutations have only small pre-I31 HDL particles with hypercatabolism ofapoA-I and have premature CHD. Patients affected with apoA-I deficiencyhave no HDL and have strikingly premature CHD. Whereas, patientsaffected with LCAT deficiency have only pre13-1 and a-4 HDL particles,and are at moderate to high risk for CVD. Different mutations in thecholesterol ester transfer protein (CETP) can cause either increased ordecreased CETP activity, resulting in different changes in HDLparticles. High CETP activity results in low levels of large a-1 andhigh levels of the small pre13-1 HDL particles. High CETP activity isassociated with significant increased risk for CVD. Low CETP activity,which may be due to mutations in the gene encoding CETP or to effects ofvarious drugs, causes high levels of a-1 HDL and low levels of pre13-1HDL. This HDL subpopulation profile (high a-1 and low pre(3-1) isassociated with protection against CVD. Various mutations in the genesencoding lipoprotein-, hepatic-, and secretory-phospholipases can alsobe detected and recognized by their specific HDL subpopulation profileusing this method.

Most importantly, the HDL subpopulation profile can differentiatesubjects with increased risk for CVD independent of the HDL-C level.This is very important, as some subjects or an entire ethnic group mayhave low HDL-C level without any history of elevated CVD risk due to thefact that these subjects have not only increased HDL catabolism, butalso enhanced HDL function. These subjects have a normal HDL particledistribution. However, some subjects with high HDL-C may experience aCVD event due to low HDL catabolism or dysfunctional HDL as seen with adefective SRB1 function.

Similar to HDL, LDL can also be separated into particles havingdifferent sizes, most commonly separated into small dense (sd) LDL andlarge LDL particles. It is proven and widely accepted in the lipoproteinfield that sdLDL-C is more atherogenic than large LDL-C. The most commonmethod for separating LDL by size is electrophoresis. The quantificationof different LDL fractions is based on lipid staining in the gel,followed by density scanning and integrating the area under the curve.The major disadvantages of this method are that it is labor and timeconsuming, and has poor resolution. A more recent method involves theuse of a specific mixture of detergents for removing other lipoproteins,and then measuring cholesterol only in small dense LDL or sdLDL. Thismethod is adaptable to high throughput automated analyzers, and it hasbeen standardized.

Risk for CVD is significantly higher in subjects with impaired glucosehomeostasis. Risk for CVD among type 2 diabetic patients is as high asthe risk among subjects with elevated LDL-C level. There are severalways to determine glucose homeostasis including the measurement offasting and post-prandial blood glucose levels, insulin levels, andhemoglobin-Alc (HbAlc) determinations. Currently, HbAlc is the mostcommonly used test to determine the severity of diabetes. The methodneeds red blood cells and fresh samples. Because the in-vivo half-lifetime of hemoglobin is about two to three months, measuring the amount ofglucose attached to hemoglobin or HbAlc has been shown to be anexcellent measure of long-term (8-12 weeks) blood glucose control.However, doctors who treat patients with CVD usually look for a shortertime period to determine whether the medications they prescribe affectdiabetes. Moreover, there is not a wide range of values in the normalpopulation. There is a way to measure shorter term changes in glucosehomeostasis, namely by measuring glycated albumin (GA) as the percentileof plasma total albumin, which represents the glycation status over thepast two to four weeks versus the three month period of HbAlc. Thismeasurement is easy; utilizes plasma samples, and can be measured fromstored (frozen) samples. Further, its value correlates well with HbAlcvalues, and due to the larger dynamic range of GA % measurement,subjects without known diabetes can be characterized more accuratelywith regard to their risk of developing diabetes and CVD. GA %measurement can also facilitate the diagnosis of pre-diabetes status.

Cardiovascular disease is considered both a lipid storage and aninflammatory disease. One of the inflammatory markers that have beenshown to be an independent marker of CVD is C-reactive protein (CRP).CVD patients have increased CRP level. CRP has been a very well studiedCVD-risk factor in the last couple of years. CRP is measured in plasmausing a high sensitivity CRP assay kit. Recently, it has been found thatCRP has several molecular forms (CRPmf) in human plasma. These formsdiffer in electrophoretic mobility and size, as assessed bypolyacrylamide gel electrophoresis and immuno-localization under specialconditions. The concentrations of the smallest molecular form (CRP mf4),or the ratio of this small CRP mf4 to the largest one (CRP mf1) ispositively associated with fat cell mass (obesity) and with the presenceof CVD.

Adiponectin is a protein hormone that modulates a number of metabolicprocesses, including glucose regulation and fatty acid catabolism.Adiponectin is exclusively secreted by adipose tissue into thebloodstream and is very abundant in plasma relative to many otherhormones. Levels of the hormone are inversely correlated with body fatpercentage in adults. The hormone plays a role in the suppression of themetabolic derangements that may result in type 2 diabetes, obesity,atherosclerosis and non-alcoholic fatty liver disease.

Despite the evident need for a better predictor of CVD, the market lacksa diagnostic solution consisting of a complete test panel for screeningof an individual's CVD risk and a process for an accurate andindividualized diagnosis and treatment plan derived from the results ofthe screening tests in order to optimize therapy and decrease CVD risk,especially in those patients who already have established CVD.

SUMMARY OF THE INVENTION

Diagnostic screening panels for assessing cardiovascular risk exist;however, these tests are limited in the breadth of CVD risk factors.

In view of the above, there is a need for a diagnostic solution thatwill provide more complete CVD-risk assessment, and thereby assure amore accurate and individualized treatment plan. Further, there is aneed for an individualized treatment protocol utilizing recentdevelopments in HDL particle subfractionation.

It is, therefore, an aspect of the present invention to provide anextended CVD-risk panel that tests and/or measures the combination oftraditional risk factors and new important risk markers.

It is another aspect of the present invention to provide a means forobtaining more detailed information about the disturbance in lipoproteinand glucose metabolisms and inflammatory status of an individual.

It is another aspect of the present invention to provide a means formonitoring drug effectiveness with greater accuracy and speed thantraditional testing methods in the treatment of cardiovascular disease.

It is another aspect of the present invention to provide a CVD protocolalgorithm for the analysis of the results obtained from the extendedrisk panel testing, in order to facilitate personalized treatmentoptions for a patient.

It is another aspect of the present invention to provide a treatmentplan for the personalized treatment of a cardiovascular disease ormanagement of cardiovascular risk in an individual.

The present invention pertains to a comprehensive diagnostic screeningsolution for assessing CVD risk. This novel diagnostic solutioncomprises an extended CVD-risk panel for testing of traditional riskfactors in combination with new and emerging tests. The extended CVDrisk assessment panel tests for the following: general metabolic factorsof blood urea nitrogen or BUN, creatinine, BUN/creatinine ratio,glomerular filtration rate (GFR), calcium, alkaline phosphatase, liverenzymes AST and ALT, creatine kinase (CK), thyroid stimulating hormone(TSH), sodium, potassium, chloride, carbon dioxide, and uric acid;specialized heart disease factors in addition to total cholesterol,total triglyceride, high density lipoprotein (HDL) cholesterol,calculated low density lipoprotein (LDL) cholesterol, non-HDLcholesterol, very low density lipoprotein (VLDL) cholesterol, and totalcholesterol/HDL cholesterol ratio; as well as specialized lipid factorsof direct LDL cholesterol, small dense LDL cholesterol, apolipoproteinapoA-I and apoB, lipoprotein (a) or Lp(a), highly sensitive C-reactiveprotein (hsCRP) and CRP molecular forms (CRPmf), lipoprotein associatedphospholipase A2 (LpPLA2), fibrinogen, glycated albumin, globulin,albumin/globulin ratio, glycosylated hemoglobin, total bilirubin,adiponectin, homocysteine, and insulin; HDL subpopulations (bytwo-dimensional gel electropheresis) of a-1 HDL, a-2 HDL, a-3 HDL, a-4HDL, and pre13-1 HDL particles; markers of cholesterol synthesis (plasmalevels of lathosterol and desmosterol); markers of cholesterolabsorption (plasma levels of beta sitosterol, campesterol, andcholestanol); and other specialized testing pertaining to apolipoproteinE and Factor V Leiden genotyping, and NT-proBNP or N-terminal pro-brainnatriuretic peptide.

This extended CVD-risk panel yields more detailed information about thedisturbance in lipoprotein and glucose metabolisms and inflammatorystatus in an individual, and about the effectiveness of an appliedmedication to treat disorders of lipoprotein metabolism, inflammation,and glucose homeostasis, thereby leading to more personalized treatment.

The diagnostic solution of the present invention provides not onlysuperior assessment of CVD risk prospectively, but also assessment ofrisk of recurrent CVD events in individuals who have already experiencedCVD events. Further, the panel of CVD-risk markers may be selected so asto predict a change in risk for CVD, as well as to optimize treatment.The CVD risk panel comprises at least one test or measurement (“test ormeasurement” shall collectively be referred to as “tests”), depending onthe particular marker, for each of the following markers: totalcholesterol, total triglyceride, lipoprotein particles, apolipoproteins,diabetes and fat metabolism, plasma sterols, inflammatory markers,genetic testing, and secondary causes of high cholesterol; said testsforlipoprotein particles comprising at least one test for each of thefollowing: direct high density lipoprotein cholesterol, HDL subparticlefractionation by two-dimensional gel electrophoresis, direct low densitylipoprotein cholesterol, direct small dense LDL cholesterol, percentageof LDL cholesterol as small dense LDL cholesterol, lipoprotein (a), andnon-HDL cholesterol and total cholesterol/HDL cholesterol ratio; saidtest for markers of diabetes comprising at least one test for each ofthe following: insulin, albumin, glycosylated hemoglobin, and glycatedalbumin; said test for plasma sterols comprising at least one test foreach of the following: lathosterol, desmosterol, campesterol,beta-sitosterol, and cholestanol; said test for inflammatory markerscomprising at least one test for each of the following: C reactiveprotein and lipoprotein associated phospholipase A2; said test forgenetic testing comprising at least one test for each of the following:apolipoprotein E genotype, and factor V Leiden genotype; and said testfor secondary causes of high cholesterol comprising at least one testfor each of the following: creatinine, blood urea nitrogen, creatinekinase, liver transaminases, alkaline phosphase, thyroid stimulatinghormone, and uric acid. Each of the tests for each of the markers may beselected so as to predict present risk and a change in risk for CVD, aswell as to optimize treatment.

The present invention also pertains to a method of personalizedtreatment of cardiovascular disease. CVD is a combined term for about 20diseases currently known. The CVD risk assessment panel of the presentinvention enables the determination of which disease of the group ofdiseases that a particular patient has or presents a risk therefor. Formany of these diseases, the particular biological or physiologicalmechanism of dysfunction is known. Further, particular useful andbeneficial therapies are known for many of these dysfunctions. Therapiesin this respect can be personalized with lifestyle therapy (modifieddiet low in cholesterol, saturated fat, trans fat, and sugars, andincreased in fiber and essential fatty acids, as well as increasedphysical activity), nutritional supplements (such as omega 3 fattyacids, and coenzyme Q10), and particular drug(s), such as effectivestatins, cholesterol absorption inhibitors, niacin products, fibrates,resins, and other medical therapies in development (including CETPinhibitors). Thus, the CVD risk assessment panel of the presentinvention allows the treating healthcare practitioner to determine thepatient's particular disease dysfunction, and to propose an optimalpersonalized treatment plan for the patient.

The present invention comprises the performance of diagnostic analysis,utilizing the CVD diagnosis protocol algorithm of the present invention,to optimally assess the CVD risk of a patient, and facilitate thepersonalized treatment plan for an individual while monitoring drugeffectiveness, with greater accuracy and speed than traditional testingmethods for the treatment of cardiovascular disease or prevalent risktherefor.

The above summary of the present invention is not intended to describeeach illustrated embodiment or every implementation of the presentinvention. The figures and the detailed description that followparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, in which:

FIG. 1A-FIG. 1C illustrate typical results of the HDL subpopulationanalysis assessed by two-dimensional gel electrophoresis,immono-localization, and image-analysis, as described in prior art.These figures serve to demonstrate the effectiveness of this testingmethod as applied to the present invention. In FIG. 1A and FIG. 1B, HDLparticles separated from whole plasma by electrophoresis are shown inthe vertical dimension for size (from large on top to small on thebottom of the gel), and in the horizontal dimension by charge intopre-B, a, and pre-a mobility. Subsequently, particles are analyzed fortheir apoA-I content by immunoblotting with specific antibodies and thenperforming quantitative image analysis. The HDL particle profile of ahealthy control subject is shown on the left (FIG. 1A) and a patientwith CVD is shown in the middle (FIG. 1B). These figures show that CVDpatients have less a-1 HDL and a-2 HDL (which are the most protectiveparticles), and more pre-B1 HDL than healthy subjects (FIG. 1A), andthat a high concentration of pre-B1 HDL is associated with higher risk.The “*” indicates the position of albumin, a 67kD plasma protein, thatmarks the a-front. The scan of the a region, as shown at the bottomportion of FIG. 1A, represents the integration of the a-mobility HDLparticles. This scan is used to define the positions of the individual amobility particles. The concentration of apoA-I in all HDL particles isthen determined via image analysis. The illustration in FIG. 1Crepresents the positions of all apoA-I containing HDL particles.

FIG. 2 illustrates non-denaturating one-dimensional gel electrophoresisof whole plasma, followed by immunoblotting with specific antibody forCRP, according to an embodiment of the present invention. The gels showndocument the presence of five individual molecular forms of CRP. The CRPpatterns of controls are shown in the top panel, while the patternsobserved in obese, diabetic patients with CVD are shown in the lowerpanel. The major difference between CVD cases and controls is thepresence of CRP molecular form 4 in the CVD cases.

FIGS. 3A-R provide a flowchart depicting the various aspects of the CVDdiagnosis and treatment protocol algorithm for performing diagnosisanalysis or diagnosis and treatment analysis, according to oneembodiment of the present invention.

FIGS. 4A and 4B depict methods for performing a diagnosis and treatmentanalysis, according to certain embodiments. FIG. 4A is a diagramgenerally depicting the method of performing a diagnosis analysisutilizing the CVD diagnosis and treatment protocol algorithm, with thehealthcare practitioner devising the treatment plan for the patient,according to one embodiment of the present invention. FIG. 4B is adiagram generally depicting the method of performing a diagnosis andtreatment analysis utilizing the CVD diagnosis and treatment protocolalgorithm, according to one embodiment of the present invention.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

The present invention pertains to a diagnostic solution comprising anextended risk assessment panel for cardiovascular disease. The presentinvention also pertains to a personalized treatment solution for thetreatment of CVD in an individual.

1. Definitions

The term “adiponectin”, as used herein, refers to a protein hormone madein a person's fat, but only in subcutaneous fat, and not in visceral fat(the fat around a person's middle portion that increases his or herwaist size). High levels of adiponectin indicate protection from heartdisease, while low levels increase CVD risk in overweight or obesesubjects.

The term “alkaline phosphatase”, as used herein, refers to a measure ofliver function and bile flow, as well as bone status. An alkalinephosphatase value between 33 U/L and 130 U/L is considered normal;whereas, an elevated value may indicate obstruction of bile flow, excessbreakdown of bone, or malignancy.

The term “a-1 HDL particle” or “a-1”, as used herein, refers to theapoA-I concentration in the HDL particles with a median size of about11.0 nm. It is one of the most important HDL particles for predictingheart disease. This large particle delivers cholesterol to the liver.This HDL particle is large and lipid-rich; it contains 6 molecules ofapoA-I, a large amount of free cholesterol and phospholipids (PL) on thesurface, and cholesterol ester and TG in the core. This is the particlethat interacts with scavenger receptor B1 (SRB1) in the liver and dumpscholesterol into the bile. A decreased level marks an inadequate HDLmetabolism and is associated with increased risk for CVD. A value below12.0 mg/d1 is associated with increased heart disease risk in men and avalue below 18.0 mg/dl is associated with increased heart disease riskin women, while a value between 12.0 mg/dl and 17.0 mg/dl in men and avalue between 18.0 mg/dl and 28.0 mg/dl in women is consideredborderline. A value above 17.0 mg/dl in men and above 28.0 mg/dl inwomen is considered normal.

The term “a-2 HDL particle” or “a-2”, as used herein, refers to theapoA-I concentration in the HDL particles having a median size of about9.20 nm. It is one of the most important HDL particles for predictingheart disease. This HDL particle is quite large and delivers cholesterolto the liver. A value below 38.0 mg/dl is associated with increasedheart disease risk in men and a value below 45 mg/d1 is associated withincreased heart disease risk in women, while a value is between 38.0mg/dl and 40.0 mg/dl in men, and between 45.0 mg/dl and 52.0 mg/dl inwomen is considered borderline. A value above 40.0 mg/dl in men andabove 52.0 mg/dl in women is considered optimal.

The term “a-3 HDL particle” or “a-3”, as used herein, refers to theapoA-I concentration in the HDL particles having a median size of about8.00 nm. There is no established optimal or normal level for a-3;however, a ratio of a-1 to a-3 of less than 0.3 is an indication ofabnormal HDL metabolism and increased risk for CVD.

The term “a-4 HDL particle” or “a-4”, as used herein, refers to theapoA-I concentration in the HDL particles having a median size of about7.42 nm. There is no established optimal or normal level for a-4;however, a ratio of a-1 to a-4 of less than 0.6 is an indication ofabnormal HDL metabolism and increased risk for CVD.

The term “albumin”, as used herein, refers to a measure of proteinmetabolism and nutritional status. An albumin value of 3.5-4.9 g/dl isconsidered normal; a decreased value may indicate malnutrition orchronic illness.

The term “apolipoprotein A-I” or “apoA-I”, as used herein, refers to themeasure of the most abundant protein component of HDL having a 28 kiloDaltons (KD) molecular weight. ApoA-I is an essential component of HDL;low level of apoA-I is associated with low level of HDL-C and high riskfor CVD.

The term “apoA-I Fractional Catabolic Rate”, as used herein, refers tothe rate of degradation of apoA-I.

The term “apolipoprotein B” or “apoB”, as used herein, refers to ameasure of the fundamental protein component of VLDL and LDL having a500 KD molecular weight. Apolipoprotein B has been reported to be abetter predictor of heart disease than LDL cholesterol. An apoB value ofabove 120 mg/dl indicates high risk, a value between 60 mg/dl and 120mg/dl is considered borderline, and a value below 60 mg/dl is consideredto be optimal. A high value is associated with an increased risk forCVD.

The term “apolipoprotein E genotyping” or “apoE”, as used herein, refersto testing of DNA for determining one of the genetic causes of heartdisease risk. There are three different forms of apoE in human plasma:apoE2, apoE3 and apoE4. The normal apoE genotype is apoE3/3, while theapoE2/2 genotype is associated with an increased risk of elevatedtriglyceride values, and the apoE4/4 and apo4/3 genotypes are associatedwith increased LDL-C, increased cholesterol absorption, and increasedrisk of CVD and dementia.

The term “ALT”, as used herein, refers to a measure of liver function.Normal ALT value is between 6 U/L and 40 U/L; a value of above 120 U/Lis definitely abnormal and indicates either a fatty liver, liverdisease, or a side effect of a medication (such as a statin).

The term “AST”, as used herein, refers to a measure of liver function.An AST value between 10 mg/dl and 35 mg/dl U/L is considered normal;whereas, a value above 105 mg/dl is definitely abnormal and indicates afatty liver, liver disease, or a side effect of a medication (e.g., astatin).

The term “beta sitosterol”, as used herein, refers to a marker ofcholesterol absorption. A beta sitosterol value above 250 in both menand women is considered very high and is diagnostic ofbeta-sitosterolemia or phytosterolemia associated with premature heartdisease, while a value above 150 in women and above 160 in men isconsidered high, and a value between 130 and 150 in women and between150 and 160 in men is considered borderline. A value below 130 in womenand below 150 in men is considered optimal. [Provided in units relativeto total plasma cholesterol as ‘mmol x 10²/mol of cholesterol’.]

The term “blood urea nitrogen” or “BUN”, as used herein, refers to ameasure of kidney function. A BUN value of 25 mg/dL or below isconsidered normal, while an elevated value may indicate decreased kidneyfunction.

The term “calculated low density lipoprotein cholesterol”, “low densitylipoprotein cholesterol” or “LDL-C”, as used herein, refers to the levelof the cholesterol in the particle that causes heart disease. This valueis calculated by subtracting the sum of HDL cholesterol andtriglyceride/5 from total cholesterol. The calculation is not valid ifthe subject is not fasting or if the triglyceride value is above 400mg/dl. An LDL-C value above 160 mg/dl is considered very high, whereas avalue between 130 mg/dl and 160 mg/dl is considered high, a valuebetween 100 mg/dl and 130 mg/dl is considered borderline, and a valuebelow 100 mg/dl is considered optimal. For heart disease patients, anideal LDL cholesterol level is below 70 mg/dl. A high value isassociated with an increased risk of CVD.

The term “campesterol”, as used herein, refers to a marker ofcholesterol absorption. A value greater than 250 in both men and womenis considered very high, a value greater than 200 in women and greaterthan 220 in men is considered high, and a value between 180 and 200 inwomen and between 200 and 220 in men is considered borderline, while avalue less than 180 in women and less than 200 in men is consideredoptimal. [Provided in units relative to total plasma cholesterol as‘mmol x 10²/mol of cholesterol’.]

The term “cholestanol”, as used herein, refers to a marker ofcholesterol absorption. A cholestanol value above 250 in both men andwomen is considered very high and is diagnostic of cerebrotendinousxanthomatosis associated with neurologic disease, a value above 140 inmen and women is considered high, a value between 130 and 140 in men andwomen is considered borderline, while a value below 130 in both men andwomen is considered optimal. [Provided in units relative to total plasmacholesterol as ‘mmol x 10²/mol of cholesterol’.]

The term “creatinine”, as used herein, refers to a direct measure ofkidney function. A creatinine value below 1.20 mg/dL is considerednormal; whereas, an elevated value indicates decreased kidney function.

The term “creatinine kinase” or “CK”, as used herein, refers to a muscleenzyme test. A creatinine kinase value between 0 and 165 U/L isconsidered normal; whereas, an elevated value, especially above 1650U/L, indicates significant breakdown of muscle, either from heavyexercise or from a medication (rarely), such as a statin. If the patienthas such an elevated value and experiences muscle aches and pains notclearly related to exercise, then the patient should stop taking thestatin.

The term “desmosterol”, as used herein, refers to a marker ofcholesterol synthesis. A desmosterol value above 80 in women and above75 in men is considered high, while a value between 70 and 80 in womenand between 70 and 75 in men is considered borderline. A value below 70in both men and women is considered optimal. [Provided in units relativeto total plasma cholesterol as ‘mmol X 10²/mol of cholesterol’.]

The term “direct LDL cholesterol”, as used herein, refers to direct lowdensity lipoprotein cholesterol. A direct LDL cholesterol value above160 mg/dl is very high, while a value between 130 mg/L and 160 mg/dl isconsidered high, a value between 100 mg/dl and 130 mg/dl is consideredborderline, and a value below 100 mg/dl is considered optimal. In heartdisease patients, an ideal LDL cholesterol level is less than 70 mg/dl.A high value is associated with an increased risk of heart disease.

The term ‘Factor V Leiden”, as used herein, refers to a genetic variantin clotting factor V that causes increased risk for the development ofclot formation in the veins of the legs, which can result in such clotsmoving to the lungs. This is a genetic test.

The term “glycated albumin”, as used herein, refers to a test todetermine the quantity of glucose attached to the albumin in a person'sblood. It is a potent predictor of heart disease in the non-diabeticpopulation, and a potent predictor of complications in those withdiabetes. A value above 16.5% indicates the presence of diabetesmellitus.

The term “HDL subfractionation by two-dimensional gel electrophoresis”,“two-dimensional gel electrophoresis”, “two-dimensional HDL separation”or “HDL fingerprinting”, as used herein, refers to a technology thatmeasures different HDL particles by directly separating the particles bysize and charge, and then measuring the amount of the protein A-I ineach particle. It assesses how well a person's HDL particles arefunctioning in order to help remove cholesterol from the body. This testnot only measures the small HDL particles that pick up cholesterol fromthe artery wall but also the large HDL particles that delivercholesterol to the liver. These small HDL and large HDL particles helpprovide very precise information about a person's heart disease risk.Also, measuring these particles helps to determine how well a therapywith medication(s) is working in a patient.

The term “healthcare facility”, as used herein, refers to a hospital,clinic, healthcare practitioner's facility, laboratory or medicaltesting or imaging facility, or the like.

The term “healthcare practitioner”, as used herein, refers to ahealthcare professional or a healthcare provider, such as a physician, anurse practitioner, or a physician's assistant, who provides or managesthe medical care of a patient. The healthcare practitioner is authorizedto conduct the testing of the patient with the extended test paneldisclosed herein, perform diagnosis analysis or diagnosis and treatmentanalysis via the diagnosis and treatment protocol algorithm disclosedherein, interpret the results of the diagnosis analysis, interpret theresults of the diagnosis and treatment analysis, devise or modify anindividualized treatment plan, monitor the efficacy of drug therapy,monitor the effectiveness of treatment plan, and manage the health careof a patient.

The term “high density lipoprotein cholesterol” or “HDL-C”, as usedherein, refers to the cholesterol measurement in plasma, after theremoval of apoB-containing lipoproteins (VLDL and LDL particles). Highlevels of HDL cholesterol, above 60 mg/dl, protect against heartdisease. A value between 40 mg/L and 60 mg/dl is considered borderline,while a low HDL cholesterol value, below 40 mg/dl in men and below 50mg/dl in women, is associated with an increased risk of heart disease.

The term “highly sensitive C Reactive Protein” or “hsCRP” or “CRP”, asused herein, refers to a measure of inflammation in a person's blood. ACRP value above 3.0 mg/L is considered high, a value between 2.0 mg/Land 3.0 mg/L is considered borderline, and a value of less than 2.0 mg/Lis considered normal, while some authorities recommend maintaining CRPvalues below 1.0 mg/L. A high value is associated with an increased riskfor CVD.

The term “highly sensitive C-reactive protein molecular form” or“CRPmf’, as used herein, refers to a measure of specific CRP protein(complex) in a person's blood. CRP mf-1 is the largest form and CRP mf-4is the smallest form of the major molecular forms of CRP. The presenceof CRPmf-4 in plasma is associated with an increased CVD risk in obeseand diabetic subjects.

The term “insulin”, as used herein, refers to a very important hormonein a person's blood that regulates a person's blood glucose. A fastinginsulin level above 20 microunits/ml (“mU/ml”) is considered high, avalue between 10 mU/ml and 20 mU/ml is considered borderline, and avalue below 10 mU/ml is considered ideal. High values are associatedwith insulin resistance and an increased risk for CVD. A very low value,below 5 mU/ml, in the setting of diabetes is consistent with insulindeficiency and a need for insulin therapy.

The term “lab technician”, as used herein, refers to an authorizedperson employed at a healthcare facility, and who may conduct thetesting of a patient with the extended panel of the present invention. Alab technician may also be authorized to enter the results of thetesting for the performance of the diagnosis analysis via the protocolalgorithm of the present invention, and/or to perform said analysis.

The term “lathosterol”, as used herein, refers to a marker ofcholesterol production. A lathosterol value above 150 in women and above135 in men is considered high, a value between 130 and 150 in women andbetween 120 and 135 in men is considered borderline, while values below130 in women and below 120 in men are considered optimal. [Provided inunits relative to total plasma cholesterol as ‘mmol x 10²/mol ofcholesterol’.]

The term “lipoprotein (a)” or “Lp(a)”, as used herein, refers to an LDLparticle with another protein (referred to as apo(a)) attached thereto.A high value of this particle can interfere with the process of breakingup clots in a person's arteries. An Lp(a) value above 30 mg/dl isconsidered high, while a borderline value is between 20 mg/dl and 30mg/dl, and an optimal value is below 20 mg/dl. A high value isassociated with an increased risk of heart disease.

The term “lipoprotein associated phospholipase A2” or “LpPLA2”, as usedherein, refers to a marker of inflammation. An LpPLA2 value above 235ng/ml is considered high, while a value between 200 ng/ml and 235 ng/mlis considered borderline, and a value below 200 ng/ml is consideredoptimal. A high value is associated with an increased risk of heartdisease.

The term “non-HDL cholesterol”, as used herein, refers to a calculatedvalue (total cholesterol minus HDL cholesterol). A non-HDL cholesterolvalue above 190 mg/dl is considered very high risk, while a valuebetween 160 mg/dl and 190 mg/dl is considered high risk, and a valuebetween 130 mg/dl and 160 mg/dl is considered borderline. A value below130 mg/dl is considered optimal, with a value below 100 mg/dl being atarget value for patients with heart disease. A high value is associatedwith an increased risk of heart disease.

The term “N-terminal pro-Brain natriuretic peptide” or “NT-proBNP”, asused herein, refers to a marker of heart disease stress. High levelshave been associated with an increased risk of mortality in patientswith heart disease. An NT-proBN value above 450 pg/dl is consideredhigh, while a value between 125 pg/ml and 450 pg/ml is consideredborderline high, and a value below 125 pg/dl is considered optimal.

The term “patient” or “subject”, as used herein, refers to a person oran individual who is at risk for or has already exhibited one or moreaspects of a disease or, particularly cardiovascular disease or CVD.

The term “pre13-1 HDL particle”, as used herein, refers to an importantHDL particle for predicting heart disease. This HDL particle is quitesmall, contains 2 apoA-I and 16 phospholipid (PL) molecules. This is theparticle that picks up cholesterol from the artery wall via the ABCA1pathway. An increased level marks an inadequate HDL metabolism and isassociated with an increased risk for CVD. A value above 20.0 mg/dl isassociated with increased heart disease risk in both men and women,while a value between 15.0 mg/dl and 20.0 mg/dl in both men and women isconsidered borderline. A value below 15.0 mg/dl in both men and women isconsidered optimal.

The term “small dense LDL cholesterol” or “sdLDL-C”, as used herein,refers to the cholesterol level in the LDL particles that confer thehighest risk of heart disease. An sdLDL-C value above 40 mg/dl isconsidered high, while a value between 20 mg/dl and 40 mg/dl isconsidered borderline, and a value below 20 mg/dl is considered optimal.In heart disease patients, it is recommended that small dense LDLcholesterol level be maintained below 20 mg/dl. A high value isassociated with an increased risk of heart disease.

The term “thyroid stimulating hormone” or “TSH”, as used herein, refersto a sensitive measure of thyroid function. A thyroid stimulatinghormone value between 0.5 micrograms/L and 5.5 micrograms/L isconsidered normal. A high value indicates an underactive thyroid glandand hypothyroidism, which causes fatigue, cold intolerance, dry skin,constipation, and an elevated blood cholesterol level, while a low valueis due to an overactive thyroid gland, and can cause a rapid heart beatand even palpitations.

The term “total cholesterol”, as used herein, refers to the sum of thecholesterol in all of the cholesterol carrying particles in a person'sblood. A total cholesterol value above 240 mg/dl is considered high,while a value between 200 mg/dl and 240 mg/dl is considered borderline,and a value below 200 mg/dl is considered optimal. A high value isassociated with an increased risk of heart disease.

The term “total cholesterol/HDL cholesterol ratio”, as used herein,refers to a measure of heart disease risk. A value above 6.0 isconsidered very high risk, while a value between 5.0 and 6.0 isconsidered high risk, a value between 4.0 and 5.0 is consideredborderline, and a value below 4.0 is considered optimal (recommended asa target by both the Canadian and European guidelines panel for heartdisease patients). A high value is associated with an increased risk forCVD.

The term “triglyceride” or “TG”, as used herein, refers to a fat in aperson's blood. Very high levels of triglyceride, above 1000 mg/dl,increase the risk of pancreatitis, while a high value, above 150 mg/dl,is associated with an increased risk for CVD.

The term “uric acid”, as used herein, refers to a substance in thebloodstream derived from the breakdown of protein. High level of uricacid, above 10.0 mg/dl, is associated with an increased risk for bothgout and CVD, and can be a cause of gouty arthritis and kidney stones.

The term “very low density lipoprotein cholesterol” or “VLDL-C”, as usedherein, refers to a lipoprotein particle in fasting plasma. A VLDL-Cvalue above 30 mg/dl is considered high, while a value below 30 mg/dl isconsidered optimal. A high value is associated with an increased riskfor CVD.

It is to be understood that the singular forms of “a”, “an”, and “the”,as used herein and in the appended claims, include plural referenceunless the context clearly dictates otherwise.

2. Diagnostic Solution for Assessing Cardiovascular Disease Risk

The present invention provides an unique diagnostic solution, in that anextensive risk assessment panel provides information pertaining to thelevels of certain important markers, as well as information pertainingto genetic testing and traditional risk factors; such knowledgefacilitates a healthcare practitioner to optimize therapy with lifestylemodification and pharmacologic therapy in subjects with cardiovasculardisease (e.g., heart disease, stroke), or individuals at high risk ofdeveloping these disorders.

2.1. The Extended Risk Assessment Panel

The diagnostic solution of the present invention comprises a rathercomprehensive test panel for assessing CVD-risk beyond the traditionalrisk factors and tests. This solution provides a comprehensive CVD riskassessment by testing or measuring (collectively, “testing”) for all ofthe following: the general metabolic factors, the specialized heartdisease factors, the specialized lipid factors, the plasma sterols, theHDL subpopulations (by two-dimensional gel electrophoresis), the CRPmolecular forms, and other specialized testing pertaining toapolipoprotein E genotyping, Factor V Leiden genotyping, NT-proBNP orN-terminal pro-brain natriuretic peptide, adiponectin, and glycatedalbumin. With the exception of genetic testing, which need only beperformed once in a lifetime for a particular patient due to the natureof the test, such as at the time of initial assessment testing, thepanel of tests may be performed multiple times for a given patient, forexample, when a treating healthcare practitioner needs to assess thepatient's current CVD risk, or assess the patient's response to therapy.Preferably, all tests are performed at each instance of testing of apatient, each of such instances of testing is referred to as “subsequenttesting”, as they are performed subsequent to the initial assessmenttesting.

The comprehensive nature of the risk assessment panel of the presentinvention does not mean that all tests are performed from the samesample from the patient or that the tests are all performed at the sametime, although, all of the tests that must be performed for a givenpatient are preferably performed in close proximity in time so that thetest results can be used in combination to comprehensively assess riskand perform diagnosis or diagnosis and treatment analysis. The “teststhat must be performed for a given patient” means all of the tests forinitial assessment, i.e., the first time that a treating healthcarepractitioner orders such tests for the patient, and means only thosetests that must be repeated for subsequent evaluations of that patient,such as the occasions when a treating healthcare practitioner needs toassess the patient's current CVD risk, or the patient's response totherapy. Certain test results can be re-utilized, without re-testing apatient (particularly genetic testing results; and possibly plasmasterol testing, depending on the patient), in combination with therepeated or subsequent test results, in the performance of the analysisthat yields the current diagnosis or current diagnosis and treatmentinformation for the subsequent testing time frame (as a patient's riskfor CVD or response to therapy may change during any period of time).

In order to assess an individual's complete CVD-risk profile, theextended CVD-risk panel is utilized to specifically test for thefollowing:

1. Total Cholesterol

2. Total Triglyceride

3. Lipoprotein Particles (Lipoproteins)

-   -   3.1 Direct High Density Lipoprotein (HDL) Cholesterol    -   3.2 HDL subpopulations (by Two-Dimensional Gel Electrophoresis)    -   3.3 VLDL Cholesterol    -   3.4 Direct Low Density Lipoprotein (LDL) Cholesterol    -   3.5 Direct Small Dense LDL Cholesterol    -   3.6 Percentage of LDL Cholesterol as Small Dense LDL Cholesterol    -   3.7 Lipoprotein (a)    -   3.8 Non-HDL Cholesterol and Total Cholesterol/HDL Cholesterol        Ratio

4. Apolipoproteins

-   -   4.1 Apolipoprotein A-I (“apoA-I”) 4.2 Apolipoprotein B (“apoB”)

5. Markers of Diabetes and Fat Metabolism

-   -   5.1 Insulin    -   5.2 Albumin    -   5.3 Glycated Albumin    -   5.4 Glycosylated Hemoglobin    -   5.5 Adiponectin

6. Plasma Sterol Analysis 6.1 Lathosterol

-   -   6.2 Desmosterol    -   6.3 Campesterol    -   6.4 Beta-sitosterol 6.5 Cholestanol

7 Inflammatory Markers

-   -   7.1 C-Reactive Protein (CRP)    -   7.2 CRP molecular forms (CRPmf)    -   7.3 Lipoprotein Associated Phospholipase A2 (LpPLA2)

8. Mortality Marker

-   -   8.1 NT-Pro Brain Natriuretic Peptide

9. Genetic Testing

-   -   9.1 Apolipoprotein E Genotype    -   9.2 Factor V Leiden Genotype

10. Secondary Causes of High Cholesterol and Safety Testing

-   -   10.1 Creatine Kinase    -   10.2 Liver Transaminases (ALT, AST)    -   10.3 Alkaline Phosphase    -   10.4 Thyroid Stimulating Hormone    -   10.5 Uric Acid    -   10.6 Blood Urea Nitrogen    -   10.7 Creatinine        The results from all of these tests enable the treating        healthcare practitioner to optimize therapy in patients with or        without established CVD.

The solution of the present invention focuses on the above-listed testsas they pertain to factors that have great impact on CVD risk.

Levels of high density lipoprotein (HDL) greatly correlate with CVDrisk. Thus, testing for and measuring HDL levels is an important aspectof assessing CVD risk. While measuring HDL levels via standard and otherspecial lipid tests, such as for direct HDL cholesterol levels, providesvaluable information, measuring HDL particle compositions, i.e.,subfractions, provides a much more accurate and complete picture of howwell HDL is functioning. The effects of many drugs on these subclassesare known, and thus knowing the level of an individual's HDL subclasseswill lead to a more targeted and personalized treatment for patients.

HDL subpopulation analysis by two-dimensional gel electrophoresisinvolves separation of lipoproteins. HDL can be separated byelectrophoretic mobility into pre13, a, and prea-mobility particles, andcan be separated by size in the range of from about 6 nanometers toabout 12 nanometers. Specifically, lipoproteins are fractionatedprimarily with respect to differences in their electrophoretic mobilityand/or size. When lipoproteins are fractionated using theelectrophoretic technique, they are separated into the fractions ofpre13-mobility HDL, a-mobility HDL, and prea-mobility HDL. FIG. 1a -FIG.1c serve to demonstrate the effectiveness, and thereby the suitabilityof use of this test as a part of the present invention. FIG. 1a -FIG. 1cshow typical results for the HDL two-dimensional gel electrophoresistest. In the example shown in FIG. 1b , a patient with CVD has less ofa-1 HDL and a-2 HDL, and more of pre13-1 HDL. High levels of a-1 and a-2are associated with low risk for CVD, while high level of pre13-1 HDL isassociated with high risk for CVD. The results of HDL subpopulationanalysis will allow the healthcare practitioner to more effectivelytreat patients with agents, such as various statin drugs and niacin, tonormalize these particles and decrease CVD risk.

Cholesterol production and absorption are important aspects of CVD risk,and thus should be assessed. People have elevated total cholesterol andLDL cholesterol for a variety of reasons. One reason is that they maketoo much cholesterol in their body, and such people are ideal candidatesfor statin treatment to inhibit cholesterol production. Other peoplehave high cholesterol and LDL cholesterol because they absorb too muchcholesterol in their intestines. Such people are ideal candidates fordiet and cholesterol absorption inhibitors. Testing of plasma sterols isa means for assessing such risk, and comprises the testing of thefollowing: lathosterol (a marker of cholesterol production); desmosterol(a marker of cholesterol synthesis); and beta sitosterol, campesteroland cholestanol (markers of cholesterol absorption). The results of suchtesting reveal whether a person is a hyper-absorber or a hypo-absorber,or is a high producer or normal producer of cholesterol.

The results from the plasma sterol testing for cholesterol absorptionand production enable the healthcare practitioner to treat elevated LDLcholesterol more effectively. This will assure the use of the mostappropriate treatment at the initiation of drug therapy: a moreeffective statin, a cholesterol absorption inhibitor, or a combinationof both. It has been shown that the use of statins not only decreasescholesterol synthesis, but also increases markers of cholesterolabsorption. This may explain why low dose statins are almost aseffective as high dose statins in reducing LDL cholesterol. Doubling thedose of a statin, on average, only increases the LDL lowering effect byabout 6%. This may also explain why adding a cholesterol absorptioninhibitor to low doses of a statin is often much more effective in LDLcholesterol control than increasing the dosage of any statin to themaximum dosage. Addition of a cholesterol absorption inhibitor to anystatin therapy usually reduces LDL cholesterol by an additional 15-18%.

The measurements of cholesterol absorption and production also enablethe diagnosis of rare disorders of cholesterol metabolism associatedwith increased heart disease risk. The analysis can identify diseases,such as phytosterolemia and cerebrotendinous xanthomatosis, both ofwhich are eminently treatable.

Genetic causes of heart disease also pose a great risk, and thus shouldbe assessed. About 20% of the population carries the apoE4 genotype andthese people have higher cholesterol absorption, higher LDL cholesterollevels, and higher heart disease risk than those who do not carry theapoE4 genotype. Apolipoprotein E genotyping is a means for assessing CVDrisk by testing a person's DNA. Knowing the apoE genotype of a patientwill enable the treating healthcare practitioner to optimize anindividual's therapy as subjects carrying the apoE4 allele are moresensitive to dietary modification and less sensitive to statins in termsof LDL-C lowering.

ApoE is found on triglyceride-rich lipoproteins and HDL, and it isimportant for liver uptake of remnant lipoproteins. ApoE's majorfunction is to serve as a ligand to LDL receptor for lipoproteinscontaining apoB and apoE or containing only apo E. The plasmaconcentration of apoE is about 10 mg/dL, and its molecular weight is34,145 Daltons. There are three different apoE forms in human plasma,which are apoE2, apoE3 and apoE4. The various forms of apoE bind to theapoE-specific cell surface receptor with different affinity, leading todifferences in clearance of the apoE containing VLDL and chylomicronremnants by the liver. ApoE3 is the common form with cysteine at residue112 and arginine at residue 158. ApoE4 is a somewhat less common form,with arginines at both residues 112 and 158. Patients with apoE4catabolize LDL at a slower rate than apoE3 patients. Apo E4 has beenassociated with increased risk of CVD and dementia. ApoE2 is the leastcommon form; patients with apoE2 catabolize LDL faster than others, butthose with the apoE2/2 genotype are at increased risk for developingdysbetalipoproteinemia or type III hyperlipoproteinemia.

Studies have shown that the presence of the genetic form apoE4, found inabout 20% of the population, increases intestinal cholesterolabsorption, liver uptake of cholesterol, LDL cholesterol, heart diseaseand dementia risk, and responsiveness to diet. The same form (ApoE4)decreases response to statins in terms of lowering LDL cholesterol.Knowledge of the patient's apoE genotype, along with the other markers,enables the healthcare practitioner to provide more effective treatmentof an individual in terms of controlling LDL cholesterol and reducingthe heart disease risk with diet and medications.

The testing of a patient with the extended panel disclosed herein may becarried out in any suitable facility, including, but not limited to, ahospital, a healthcare clinic or facility, a healthcare practitioner'spractice facility, or a laboratory or medical testing or imagingfacility. The testing may be carried out at a variety of suitablefacilities, depending on the test, and the test results from eachfacility are delivered or transmitted for evaluation or analysis thatwill be performed utilizing the test results from all of the facilitiesfor a given extended panel testing. The testing may be conducted by ahealthcare practitioner, a nurse, or a lab technician. The results ofthe testing may be stored at one or more local storage facility, one ormore remote storage facility, or any combination thereof. The results ofthe testing may be accessed from one or more such storage facilities forthe performance of the diagnosis or diagnosis and treatment analysis.Preferably, the results are stored in digital format. The digital testdata may be submitted for analysis, preferably via a communicationbetween each storage facility and the processing device in communicationwith the application embodying the CVD diagnosis and treatment protocolalgorithm.

In one embodiment of the present invention, cardiovascular risk isassessed, via the use of the extended risk panel of the presentinvention, by testing the general metabolic factors, specialized heartdisease factors, specialized lipid factors, and new important factorsassociated with CVD risk, such as HDL particles by two-dimensional gelelectrophoresis, sdLDL-C, cholesterol synthesis and absorption markers,inflammatory markers, glucose homeostasis markers, and NT-proBNP.

In one embodiment of the present invention, the risk of recurrent CVDevents in individuals who have already experienced one or more CVDevents is assessed. These patients usually have very low level of a-1HDL particle levels. The risk for recurrent or new CVD event isincreased when the concentration of a-2 HDL is also significantly lowerthan normal.

In one embodiment of the present invention, the efficacy of drugtherapy, such as lipid-altering medications, is evaluated. The result ofthe efficacy evaluation is utilized in the planning of a morepersonalized treatment. The effect(s) of a given drug on CVD riskreduction is monitored based on established information, particularlythe established information that the HDL subpopulation profile is asignificant and sensitive CVD-risk marker and superior to HDL-C in riskassessment and that the different lipid lowering medications havevarious effects on the HDL subpopulation profile. In one embodiment ofthe present invention, the effect of a given drug on the LDL-C, sdLDL-C,HDL-C, TG, LpPLA2, and CRP levels, as well as on the HDL subpopulationprofile, and cholesterol synthesis and cholesterol absorption markers,is monitored.

2.2. The CVD Diagnosis and Treatment Protocol Algorithm

A patient's diagnosis and treatment plan for a cardiovascular diseaserelated disorder or risk are determined via the analyses performed inaccordance to the CVD diagnosis and treatment protocol algorithm of thepresent invention, aspects of which are illustrated in the flowchartdepicted in FIGS. 3A-3R. The present algorithm has been developed toassist the healthcare practitioner, as well the patient, in themanagement of the patient's healthcare. The algorithm addresses allrelevant factors necessary to make a comprehensive evaluation, andfacilitates an accurate diagnosis based on statistical data accumulatedfrom broad range of studies. The general method for personalizeddiagnosis and treatment of a patient utilizing this algorithm isdepicted in FIG. 4. Utilization of such an algorithm to perform ananalysis for diagnosis or diagnosis and treatment of a CVD or risktherefor not only provides a more accurate diagnosis and personalizedtreatment plan, but also makes the process quicker than the traditionalanalysis. The savings of time would not only lead to earlier treatmentof a patient, but also would provide potential cost savings (e.g., tothe patient, the insurance companies, the medical facilities, and/or thehealthcare practitioner). Further, use of such an algorithm could leadto standardizing the practice, and hence reduce likelihood ofmisdiagnosis.

The flowchart of FIGS. 3A-3R is a simplified representation of the pathsor the stepwise iterations of the diagnosis analysis performed to yielda personalized diagnosis or a personalized diagnosis and treatment planfor a patient. For the formulation of the diagnosis, the analysiscomprises a series of iterative steps, each successive step evaluateseach new data (i.e., result of a test from the extended risk panel) incombination with all data previously submitted in the previous steps inthe cycle and relevant information about the patient initiallysubmitted, (said evaluation being performed in comparison to statisticaldata included as part of the algorithm to enable this process), untilall test data entered or submitted for analysis are evaluatedcomprehensively. Upon the completion of the final step, the analysisfunction terminates, and the diagnosis result is formed upon thecompletion of the analysis function. The present invention alsocontemplates the modification or update of statistical data, which isincluded as part of the algorithm, with current statistical data as suchdata becomes available and would serve to improve the accuracy of thediagnosis and treatment results. For the formulation of the treatmentplan, the analysis comprises a series of successive iterative steps, asin the diagnosis analysis, but continues the iterative steps to evaluatethe diagnosis data in combination with other data entered in previoussteps and the relevant data about the patient initially submitted, andin view of the statistical data.

The diagnosis and treatment protocol algorithm of the present inventionmay be embodied in any suitable application, such as a computer programor code, that can facilitate its use; said algorithm or the applicationembodying said algorithm may be stored in a hard-drive of a computer(internal or external), a portable drive or disc, a server, a temporaryor permanent memory device, or any other storage means that canfacilitate the use of the algorithm and/or the results derived from theuse thereof. The algorithm (or the application embodying it) may bedistributed, gratis or for compensation, to other healthcarepractitioners or healthcare facilities, preferably to expand its use forthe benefit of greater number of patients. The algorithm or theapplication is preferably in communication with at least one processingdevice that facilitates the diagnosis analysis or diagnosis andtreatment analysis, and which may be, for example, a computer or networkprocessor. The algorithm or the application that embodies it may beaccessed locally (e.g., on a single or networked computer) or remotely(e.g., web-based network via the internet, or via an intranet). Thisaccess to the algorithm or the application may be facilitated via theuse of any suitable equipment, including, but not limited to, acomputer, an internet appliance, telephonic device, a wireless device,and the like. Access to or the use of the algorithm or the applicationembodying said algorithm or the results obtained from the use of thealgorithm may be limited or secured from general access or use, e.g.,via a password, encryption, biometric or voice-activation, or anysuitable protection or security means. The algorithm of the presentinvention may be accessed by any authorized party, e.g., a healthcareprofessional or lab technician. A patient's personal information, whichincludes but not limited to, name, address, age, contact information,and previous medical history, and/or clinical data, for example, resultsof the extended panel testing and relevant information about thepatient, may be entered or submitted, locally or remotely forprocessing, said processing includes the performance of the diagnosisanalysis or diagnosis and treatment analysis. The results from saidprocessing may be obtained by, or delivered or transmitted to, anauthorized party, e.g., a healthcare professional, a healthcare facilityor employees thereof, the patient or one who is acting on behalf of thepatient, or patient's health insurance company; said obtaining ordelivery or transmission may be performed locally or remotely; and, saidresults may be in digital, print or any other suitable format, and maybe protected or secured via any suitable means. The delivery ortransmission of the results may be automated, for example, with respectto delivery or transmission time and to the authorized parties forreceipt thereof. The results may be delivered or transmitted via anysuitable means, including, but not limited to, the Internet, anintranet, an electronic health record or management interface, telephone(land line, wireless, or VOIP), e-mail, facsimile, postal mail or inperson. A patient's confidential information, such as the results of theextended testing and/or their analysis, any of the private informationof a patient, results of other testing, and/or healthcare practitioner'snotes and prescriptions, may be coded for delivery or transmission inorder to preserve the confidentiality of such information. Said codingof patient information may be carried out in addition to any security orprotection measures utilized for delivery or transmission of suchinformation.

3. Method for the Individualized Treatment of Cardiovascular Diseases

The method of the present invention pertains to devising anindividualized treatment plan based on the results of the diagnosticanalysis performed utilizing the data obtained from the extended riskassessment panel testing. Such a treatment plan will ensure a moreaccurate and efficacious treatment of each individual. The“comprehensive” nature of the extended test panel also means thatresults from the extended panel testing are utilized in combination todiagnose a patient and devise a personalized treatment plan for thatpatient. The synergistic effect achieved from the use of the combinedtest results is far more superior, particularly with respect toaccuracy, to diagnosis made based on results from only a limited set oftests, such as the tests for traditional risk factors.

The individualized treatment plan may address treatment of an existentcardiovascular disease, reduction of the risk of developing acardiovascular disease, or a combination, in order to best manage thehealth care of an individual. The individualized treatment plan maycomprise one component, for example, dietary restriction, or increasedexercise activity, or single-agent drug therapy, or comprise multiplecomponents, for example, dual-agent (combination) drug therapy or fishoil and single-agent drug therapy.

The methods of the present invention involve the use of an extended CVDrisk assessment panel of the present invention to test a patient. Then,the results of said tests, in combination with relevant informationabout the patient, which include, but not limited to, gender, status asa smoker, diabetic, and obese, and liver, renal, and thyroiddisfunctions, are utilized with the CVD risk diagnosis and treatmentprotocol algorithm of the present invention. The relevant informationthat is available for use herein may differ from patient to patient asto type/content of information and the extent of detail. In oneembodiment of the present invention, the test results are entered intoan application embodying said algorithm to facilitate processing andanalysis thereof, to determine the cause of the patient's disorder orrisk for CVD, which may be, for example, a lipid disorder, inflammatorystress, homozygous and heterozygous apolipoprotein A-I deficiency, ABCA1deficiency, LCAT deficiency, CETP deficiency, phytosterolemia orcerebrotendinous xanthomatosis.

In one embodiment of the present invention, a healthcare practitionerutilizes the CVD risk protocol algorithm to perform diagnosis analysisutilizing the results of the complete extended CVD testing, saiddiagnosis analysis being performed comprehensively (i.e., the diagnosisis made based on all results from the complete extended risk panel andrelevant patient information), interprets said results of the analysis,and devises a treatment plan personalized to the patient based on saidinterpretation of diagnosis analysis. In one embodiment, the diagnosisanalysis is performed as an initial assessment of a patient, wherein thecomplete risk assessment panel is carried out for the patient. Inanother embodiment, the diagnosis analysis is performed as a subsequentassessment of a patient, wherein all tests except for genetic testing ofthe extended risk panel are carried out at a time period subsequent tothe initial assessment testing; however, the diagnosis analysis isperformed utilizing the results of this subsequent testing and theresults of the initial genetic testing. In another embodiment, thediagnosis analysis is performed as a subsequent assessment of a patient,wherein all tests except for genetic testing and testing for plasmasterols of the extended risk panel are carried out at a time periodsubsequent to the initial assessment testing; however, the diagnosisanalysis is performed utilizing the results of the this subsequenttesting and the results of the initial genetic testing and testing forplasma sterols. In one embodiment of the present invention, thehealthcare practitioner utilizes the results of the diagnosis analysis,in combination with relevant patient information, to devise apersonalized treatment plan for the patient, as illustrated in FIG. 4a .In one embodiment, the healthcare practitioner implements thepersonalized treatment plan for the patient, said implementationincludes, but is not limited to, prescription of lifestyle modification,prescription of one or more drugs, or a combination thereof. In oneembodiment of the present invention, a lab technician utilizes the CVDrisk protocol algorithm to perform diagnosis analysis utilizing theresults of the extended CVD testing, and delivers the results of theanalysis to a healthcare practitioner who interprets the diagnosisanalysis results, and devises a treatment plan personalized to thepatient based on the said interpretation of diagnosis analysis. In oneembodiment, a healthcare practitioner utilizes the CVD risk protocolalgorithm to perform diagnosis and treatment analysis utilizing theresults of the extended CVD testing, as illustrated in FIG. 4b . In oneembodiment, the healthcare practitioner implements the treatment planresulting from the performance of the CVD risk protocol algorithm. Inanother embodiment, the healthcare practitioner modifies the treatmentplan obtained from the performance of the CVD risk protocol algorithmand implements the modified plan for the patient. In one embodiment ofthe present invention, a healthcare practitioner monitors theeffectiveness of one or more aspects of the personalized treatment planon a patient. The healthcare practitioner may modify the personalizedtreatment plan based on the patient's response thereto, which may be,but is not limited to, inadequate effectiveness on one or more aspectsof the CVD or CVD risk, undesirable or intolerable side effect(s), or acombination thereof. The monitoring may be conducted over a short terme.g., weeks, or long term, e.g., months or years, of time; themonitoring may be conducted in recurring periods of time in a givenshort term or long term time period. The patient may be re-tested withthe present extended testing panel during the course of monitoring theeffectiveness of the personalized treatment plan, said course ofmonitoring may extend to the remainder of the patient's life.

The present invention provides certain unique features not availablewith currently existing tests for CVD risk assessment, said featuresinclude a comprehensive CVD risk assessment and individualized treatmentplanning. Further, the use of the extended risk panel and the CVD riskprotocol algorithm disclosed herein to diagnose a patient based oncomprehensive data obtained from the extended testing providessynergistic advantages over the use of limited test panels, in terms ofefficiency, greater accuracy of diagnosis, cost savings, and theshortened period of time to diagnose and treat a patient according to apersonalized plan. These advantages of the present invention also remainwhen compared to diagnosis made singly (versus comprehensively, as withthe methods of the present invention) based on results obtained from theuse of a limited test panel, even if several different limited testpanels are used (thus, resulting in multiple singly made diagnosis, andpotentially conflicting or inaccurate diagnosis), particularly when suchtesting is conducted at varying periods of time, and even if the severallimited test panels used would cover all of the tests included in theextended risk panel of the present invention. The inclusion of HDLsubpopulation analysis by two-dimensional gel electrophoresis, CRPmolecular forms, sdLDL-C, and plasma sterol testing in the extended riskpanel particularly contributes to said uniqueness. The various aspectsof the present invention are described hereinbelow in sections 3.1-3.3;the information pertaining to test results that is disclosed in thesesections is utilized in combination to make a comprehensive diagnosisand create an individualized treatment plan for a patient. Further,information in sections 3.1-3.3 contain statistical data that forms thebasis for comparison of a patient's test results thereto, said databeing provided as a part of the algorithm to facilitate the performanceof diagnosis analysis or diagnosis and treatment analysis.

3.1. Use of HDL Information

The methods of diagnosis and treatment of the present invention utilizethe findings of the Adult Treatment Panel III (ATP III) of the NationalCholesterol Education Program (NCEP-ATP III) [as published in 2001:“Executive Summary of The Third Report of The National CholesterolEducation Program (NCEP) Expert Panel on Detection, Evaluation, AndTreatment of High Blood Cholesterol In Adults (Adult Treatment PanelIII). Expert Panel on Detection, Evaluation, and Treatment of High BloodCholesterol in Adults”, JAMA, 2001, May 16; 285(19):2486-97; with anupdate published in 2004: “Implications of Recent Clinical Trials forthe National Cholesterol Education Program Adult Treatment Panel IIIGuidelines”, Grundy, S. M., et. al.; National Heart, Lung, and BloodInstitute; American College of Cardiology Foundation; American HeartAssociation. Circulation, 2004, Jul. 13; 110(2):227-39]. Accordingly,the primary goal of lipid modifying therapy is to achieve specified LDLcholesterol targets. Means of achieving this goal include therapeuticlife style changes (TLC) and drug therapy with a statin. If additionallipid parameters need modification, then agents such as ezetimibe,niacin, resins, fibrates, and fish oil preparations are used. Thesecondary goal of lipid modifying therapy is to achieve specified TG andHDL targets. Means to achieve this goal include the use of therapeuticagents, preferably, one or more fibrates, nicotinic acid, and fish oil.

It has been established that a level of HDL-C that is less than 40 mg/dLin men and less than 50 mg/dL in women is considered low; an HDL-C riseof lmg/dL has been associated with a 1-2% reduction in CVD events; andan optimal level of HDL cholesterol is greater than 60 mg/dl in both menand women. However, HDL particle analysis provides greater precision inCHD risk prediction. A 26% reduction in CVD events is observed when thelarge a-1 HDL apoA-I particle is increased by 1 mg/dl. The presentinvention involves “HDL fingerprinting”—the analyses of HDL apoA-I subparticles with the two-dimensional gel electrophoresis method, asdescribed above, and the evaluation of the data acquired therefrom tooptimize treatment as outlined below.

-   1. The present invention utilizes the information from HDL    fingerprinting in the following manner:    -   a. In patients with low HDL-C(<40 in men, <50 in women): Low        HDL-C is a significant CVD risk factor. Knowledge about HDL        fractions provides information on HDL particle distribution and        enables more precise CHD risk assessment than HDL-C measurement        alone. Moreover, HDL fingerprinting provides indicators to        enable a more precise determination of which specific treatment        option may be most effective in optimizing HDL particle        distribution. Without the information gathered from HDL        fingerprinting, the recommended therapy for all patients,        regardless of any unique circumstances of any given patient,        would be to increase the HDL-C level. The HDL fingerprinting        information adds another dimension to a patient's therapy that        greatly increases their likelihood of receiving successful        therapy; as such therapy would be individualized for that        patient.    -   b. In patients with optimal HDL-C: Patients with optimal HDL-C        level may have increased level of the small, lipid-poor pre13-1        HDL particle and decreased level of the large lipid-rich HDL sub        particle. Both of these patterns indicate an inadequate HDL        metabolism and increased risk for cardiovascular disease. The        ratio of the most athero-protective (a-1) to the less        athero-protective (a-3, a-4, and pre(3-1) HDL sub particles        indicates whether HDL function is normal or abnormal.-   2. The present invention utilizes the following information    regarding five of the apoA-I-containing HDL sub fractions (a-1, a-2,    a-3, a-4 and pre(3-1) in the determination of treatment:    -   a. a-1: a low a-1 level is the best indicator for cardiovascular        risk in patients who have no previous history of cardiovascular        events.    -   b. a-2: a low a-2 level is the best indicator of cardiovascular        risk in patients with a past history of cardiovascular events.    -   c. a-3: a low a-1 to a-3 ratio (<0.3) indicates insufficient HDL        metabolism and reflects a triglyceride metabolism problem.    -   d. a-4: a low a-1 to a-4 (<0.6) ratio indicates insufficient HDL        metabolism and reflects a cholesterol esterification problem.    -   e. pre13-1: a high pre13-1 level (greater than (“>”) 20 mg/dl)        is associated with increased risk for cardiovascular disease.        The ratio of a-1 to pre13-1 is the bestindicator of HDL        function; a decreased ratio (<0.5) reflects an increased        cholesteryl ester transfer protein (CETP) activity or imbalance        between lecithin:cholesterol acyltransferase (LCAT) and CETP        activity.-   3. The following represent, but not limited to, various diagnostic    and treatment embodiments of the present invention:    -   a. Test Results: An HDL fingerprint, marked with high levels of        a-1 HDL and a-2 HDL together with low levels of pre13-1 HDL, a-3        HDL, and a-4 HDL.        -   Diagnosis: Decreased CVD risk in the vast majority of cases.        -   Treatment: None necessary.    -   b. Test Results: No apoA-I present in plasma.        -   Diagnosis: ApoA-I deficiency.        -   Treatment: Limited to infusion of HDL mimetic, such as            pre13-1 HDL, or similar apoA-I compounds that may promote            cellular free cholesterol efflux to reduce risk for CVD.        -   Additional treatment: Optimize other risk factors, such as            smoking, high blood pressure, diabetes, LDL-C, TG, and CRP.    -   c. Test Results: ApoA-I is present only in pre13-1 HDL.        -   Diagnosis: ABCA1 deficiency (Tangier disease).        -   Treatment: Optimize other risk factors (as stated            hereinabove).    -   d. Test Results: Very low levels of apoA-I (<20 mg/dl) and        HDL-C(<10 mg/d1), and apoA-I present in pre13-1 HDL and a-4 HDL.        -   Diagnosis: Lecithin cholesterol acyltransferase (LCAT)            deficiency.        -   Treatment: Optimize other risk factors (as stated            hereinabove) and monitor for renal insufficiency and anemia.    -   e. Test Results: Low levels of HDL-C(<25 mg/dl) and apoA-I (<70        mg/dl) and very low levels of the large a-1 (<5%) and a-2 (<25%)        HDL particles, but normal levels of pre13-1, a-4, and a-3 HDL        particles.        -   Diagnosis: Can be heterozygous apoA-I deficiency, ABCA1            deficiency, or LCAT deficiency (different familial forms of            low HDL).        -   Treatment: Optimize all other risk factors (as stated            hereinabove), and increase HDL apoA-I level with the use of            niacin.    -   f. Test Results: There is only preI3-1 HDL and low levels of        amorphous a-HDL;        -   TG level greater than 1000 mg/dL.        -   Diagnosis: Lipoprotein lipase (LPL) deficiency.        -   Treatment: Restrict fat intake (<15% of calories from fat),            but recommend a diet enriched in essential fatty acids.    -   g. Test Results: Low level of HDL-C(<40 mg/d1 in men, <50 mg/d1        in women), low level of a-1 HDL (<13 mg/d1 in men, <20 mg/d1 in        women), high level of TG (>150 mg/dl) and high level of preI3-1        HDL (>15 mg/dl).        -   Diagnosis: This pattern is frequently seen and indicates            increased CETP activity. Increased CETP activity can be the            result of high triglyceride-rich lipoproteins that is the            primary substrate for CETP.        -   Treatment: As follows:        -   I. The first choice is to recommend lifestyle changes            (weight loss if obese, diet and exercise) as well as            optimizing other risk factors (as stated hereinabove). HDL            profile typically can be improved more with significant            weight loss than with any statin mono-therapy.        -   II. The second choice is therapy with a statin, to decrease            LDL-C and TG levels, increase HDL-C level, and shift the HDL            subpopulation profile beneficially (decreasing pre-(3 1 and            increasing a-1). Statin effectiveness, in order to change            the HDL particle profile beneficially, from least effective            to most effective, is: fluvastatin (adverse effect),            lovastatin (almost no change), pravastatin, simvastatin,            atorvastatin and rosuvastatin. Rosuvastatin, atorvastatin,            and simvastatin are the most effective statins in decreasing            order for increasing the large a-1 HDL level and decreasing            the small dense LDL level. However, there is a considerable            variation in individual response. Moreover, despite the            higher LDL-C decreasing capacity of 80 mg/day atorvastatin            versus the 40 mg/day atorvastatin, the higher dose has less            capacity for increasing the a-1 HDL level than the lower            dose.        -   III. If the LDL cholesterol goal (i.e., <70 mg/d1 in heart            disease patients) is not achieved, then, increase the statin            dose, or use a more effective statin, or add ezetimibe.        -   IV. If LDL goal is reached but HDL-C and a-1 HDL levels are            low, then, statin-niacin combination therapy is recommended.            Niacin increases apoA-I production and the levels of HDL-C,            apoA-I, and a-1 HDL particles, while decreasing the levels            of TG and pre13-1 HDL particles.        -   V. If LDL goal is not reached with a potent statin, a            statin-ezetimibe combination therapy is recommended. Statin            decreases cholesterol synthesis in the liver and ezetimibe            decreases cholesterol absorption in the intestine.        -   VI. In case of high level of cholesterol synthesis markers            (lathosterol and desmosterol), statin therapy is the best            choice.        -   VII. In case of a high level of cholesterol absorption            markers (elevated campesterol and(3-sitosterol), the            statin-ezetimibe combination treatment is the best option.    -   h. Test Results: Low a-1 HDL level, and normal TG and LDL-C        levels.        -   Diagnosis: Likely a genetic disorder.        -   Treatment: Niacin therapy to increase apoA-I production.    -   i. Test Results: Normal to high HDL-C level, very high a-1 HDL        level, and symptoms of CVD.        -   Diagnosis: Possible problem with scavenger receptor B1            (SRB1) mediated uptake of HDL by the liver due to decreased            scavenger receptor B1 function. HDL-mediated reverse            (direct) cholesterol transport or HDL remodeling is slowed            by decreased SRB1 function.        -   Treatment: There is no specific medication available yet for            increasing SRB1 function; however, this invention            contemplates the use of a specific medication for increasing            SRB1 function if and when such a medication becomes            available. Due to a lack of a specific medication, current            recommendation is aggressive lipid lowering strategy. No            medication is necessary to further increase HDL-C or a-1 HDL            levels.    -   j. Test Results: Normal to moderately increased TG level, normal        LDL-C level, and slightly decreased HDL-C level, but low a-1 HDL        and high pre13-1 HDL levels.        -   Diagnosis: Isolated hypertriglyceridemia.        -   Treatment: Fish oil, and/or niacin therapy. Medication with            a fibrate, such as gemfibrozil, can be an alternative            treatment.    -   k. Test Results: Level of hsCRP greater than 3 mg/L and less        than 10 mg/L.        -   Diagnosis: Increased heart disease risk.        -   Treatment: Statin or a salicylate therapy to reduce hsCRP            levels to less than 2 mg/L.    -   l. Test Results: Level of hsCRP greater than 10 mg/L and low a-1        HDL level.        -   Diagnosis: The patient may have an acute infection, and            testing should be performed again at a later date.-   4. Specific effects of pharmacological agents are as follows:    -   a. Statins decrease cholesterol synthesis, lower Tg-rich        lipoprotein (TRL) apoB and LDL apoB levels by increasing their        FCR (fractional catabolic rate), have little effect on HDL        apoA-I and apoA-II metabolism, but increase large HDL particle        level by lowering TRL, and decreasing cholesteryl ester transfer        from HDL to TRL. The best statins to increase a-1 HDL and        decrease pre13-1 HDL levels (in the order of most beneficial to        least beneficial) are        rosuvastatin>atorvastatin>simvastatin>pravastatin>lovastatin>fluvastatin.        Rosuvastatin and atorvastatin also significantly decrease        pre13-1 HDL level by decreasing CETP activity, but have little        or no effect on apoA-I kinetics.    -   b. Ezetimibe inhibits intestinal cholesterol absorption, and is        particularly effective in patients who are hyper-absorbers        whether they are off therapy or on statin therapy. Ezetimibe is        very useful for lowering LDL-C when added to statin therapy.        This drug is also effective in patients with phytosterolemia who        have inherited defects resulting in excess absorption of plant        sterols.    -   c. Resins, such as colestipol, are useful for lowering LDL-C        levels when added to statin therapy.    -   d. Fibrates increase levels of HDL-C(by 4-6%) and a-3 HDL (by        4-6%), mainly increase the level of apoA-II, increase gene        expression of apoA-I, apoA-II, LPL; but fibrates also enhance        apoA-I fractional catabolic rate (FCR), and therefore have        little effect on apoA-I concentration. Fibrates are very useful        in lowering the elevated level of triglycerides, as well as        decreasing CVD risk in those with elevated insulin levels.    -   e. Niacin increases HDL and large HDL particles by increasing        apoA-I production, lowers TRL and apoB levels by causing an        increased FCR, and also markedly raises adiponectin level. The        use of the combination of niacin and simvastatin was found to        increases a-1 HDL by 115% (in the HDL Atherosclerosis Treatment        Study population—increases in this fraction were significantly        and inversely associated with less progression in coronary        artery stenosis [“Change in a-1 HDL concentration predicts        progression in coronary artery stenosis”, Asztalos, B. F., et.        al., Arterioscler Thromb Vasc Biol., 2003, 847-852]). Niacin        therapy is ideal in combination with statin therapy in CHD        patients who have low HDL-C and a-1 HDL particle levels.    -   f. Omega 3 fatty acids, as found in fish oil preparations, are        useful in lowering triglycerides, and also in improving HDL        particle profiles to reduce CVD risk.

3.2. Use of Plasma Sterol Information

The methods of diagnosis and treatment of the present invention utilizethe information gathered from testing of plasma sterols. In oneembodiment of the present invention, plasma sterol information is usedfor diagnosis and treatment in the following 25 manner:

-   -   Test Results: Elevated cholesterol synthesis marker.    -   Diagnosis: Over production of cholesterol in patients who have        high levels of lathosterol (please refer to Table 1 and Table 2        below).    -   Treatment: High dose of an effective statin is ideal.

In one embodiment of the present invention, plasma sterol information isused for diagnosis and treatment in the following manner:

-   -   Test Results: Elevated cholesterol absorption markers in        patients who have high or very high levels of B-sitosterol and        campesterol (please refer to Table 1 and Table 2 below).    -   Diagnosis: High dietary-cholesterol absorption in the gut, which        is usually accompanied with low cholesterol synthesis.    -   Treatment: If the LDL cholesterol is not at goal, then add        ezetimibe to decrease cholesterol absorption. Ezetimibe is best        used in combination with a statin but can also be used as        monotherapy or in combination with any other lipid modifying        agent, such as a niacin or a fibrate.

In one embodiment of the present invention, plasma sterol information isused for diagnosis and treatment in the following manner:

-   -   Test Results: Elevated lathosterol (>150 moles×10² mol of        cholesterol) and desmosterol values (>80 moles×10² mol of        cholesterol), which indicate increased cholesterol levels.        Elevated beta-sitosterol (>160 moles×10² mol of cholesterol) and        campesterol (>300 moles×10² mol of cholesterol) values, which        indicate increased absorption of cholesterol.    -   Diagnosis 1: Phytosterolemia, due to over absorption of the        plant sterols beta sitosterol and campesterol. It is diagnosed        by the finding of beta-sitosterol value greater than 250        moles×102 mol of cholesterol and campesterol value greater than        400 moles×102 mol of cholesterol. Patients with this diagnosis        develop premature heart disease.    -   Treatment 1: Primary treatment is aggressive statin therapy. If        the patient is not at his or her LDL cholesterol goal while on        statin therapy, then ezetimibe, an inhibitor of both cholesterol        and plant sterol absorption, should be added to the regimen.        Therapy with an ezetimibe can also be used as a primary        treatment.    -   Diagnosis 2: Cerebrotendinous Xanthomatosism, which is due to        abnormal bile acid metabolism, is diagnosed by the finding of        cholestanol level greater than 250 moles×10² mol of cholesterol.        Patients with this diagnosis have tendinous xanthomas, and        develop neurologic diseases if their cholestanol level is not        well controlled.    -   Treatment 2: Therapy with 250 mg of chenodeoxycholate, orally        administered three times a day (total daily dose of 750 mg).

TABLE 1 Sterol Reference Values in Women Synthesis markers Absorptionmarkers Women Lathosterol Desmosterol B-sitosterol CampesterolCholestanol Optimal <130 <70 <130 <180 <130 Borderline 130-150 70-80130-160 180-300 130-160 High >150 >80 >160 >300 >160 Veryhigh >200 >150  >250 >400 >250 (Sterol values in moles × 10² mol ofcholesterol) (The symbol <signifies ‘less than’, and the symbol>signifies ‘greater than’)

TABLE 2 Sterol Reference Values in Men Synthesis markers Absorptionmarkers Men Lathosterol Desmosterol B-sitosterol Campesterol CholestanolOptimal <120 <70 <150 <200 <130 Borderline 120-135 70-75 150-160 200-220130-140 High >135 >75 >160 >300 >160 Very high >200 >150  >250 >400 >250(Sterol values in moles × 102 mol of cholesterol) (The symbol <signifies‘less than’, and the symbol> signifies ‘greater than’)

3.3. Use of CRP Molecular Form Information

The methods of diagnosis and treatment of the present invention utilizethe information gathered from gel electrophoresis testing. Table 3(below) illustrates the distribution of CRPmf in male subjects. Thosepatients with clinical signs of CVD or diabetes (DM) have higher levelsof CRPmf-4 than patients without clinical signs of CVD or diabetes.However, subjects with metabolic syndrome have the highest level ofCRPmf-4. The presence of the CRP molecular form-4 being associated withan increased CVD risk in subjects who are obese and/or diabetic, isfurther illustrated in FIG. 2 (showing CRP molecular forms forrepresentative male populations, those who are normal (indicated as“control”, no clinical signs of CVD) and those who are obese anddiabetic with clinical signs of CVD.

In one embodiment of the present invention, where CRPmf-4 is present inan obese patient, aggressive weight loss is recommended. In anotherembodiment, where CRPmf-4 is present in a diabetic patient, statintherapy is recommended for reducing LDL cholesterol and TG levels. Inanother embodiment, where CRPmf-4 is present in a diabetic and obesepatient, weight loss and statin therapy is recommended.

TABLE 3 Percent Distribution of The Most Common CRP Molecular Forms inDifferent Male Populations N = 40 in each group CRPmf-1 CRPmf-2 CRPmf-3CRPmf-4 Male control 31 ± 15 35 ± 7 28 ± 7  7 ± 11 [30] Male CVD 26 ± 1440 ± 6 20 ± 6 15 ± 14 [57] Male CVD + DM 21 ± 13 32 ± 7 26 ± 9 22 ± 16[77] Metabolic 22 ± 14 30 ± 7 25 ± 7 21 ± 13 [100] syndrome, no CVD(Values were calculated as percent distribution and presented as mean ±SD) (Numbers in [ ] represent the percentile of those subjects whohave >5% of CRPmf-4)

As noted above, the present invention pertains to an extendedcardiovascular disease risk assessment panel for testing and measuringthe combination of traditional risk factors and new important riskfactors, and to methods for personalized diagnosis and treatmentutilizing a CVD diagnosis and treatment protocol algorithm and theresults of the extended risk assessment testing. The present inventionshould not be considered limited to the particular embodiments describedabove, but rather should be understood to cover all aspects of theinvention as fairly set out in the appended claims. Variousmodifications, equivalent processes, as well as numerous structures towhich the present invention may be applicable will be readily apparentto those skilled in the art to which the present invention is directedupon review of the present application. The claims are intended to coversuch modifications.

We claim:
 1. A CVD risk panel, comprising a) at least one test for totalcholesterol, b) least one test for total triglyceride, c) at least onetest for lipoprotein particles, said at least one test for lipoproteinparticles being for at least one test for each of the following: directhigh density lipoprotein cholesterol, HDL subparticle fractionation bytwo-dimensional gel electrophoresis, direct low density lipoproteincholesterol, direct small dense LDL cholesterol, percentage of LDLcholesterol as small dense LDL cholesterol, lipoprotein (a), and non-HDLcholesterol and total cholesterol/HDL cholesterol ratio, d) one test forapolipoproteins, e) at least one test for markers of diabetes, said atleast one test for markers of diabetes being for at least one test foreach of the following: insulin, albumin, glycosylated hemoglobin, andglycated albumin, f) at least one test for plasma sterols, said at leastone test for plasma sterols being for at least one test for each of thefollowing: lathosterol, desmosterol, campesterol, beta-sitosterol, andcholestanol, g) at least one test for inflammatory markers, said atleast one test for inflammatory markers being for at least one test foreach of the following: C reactive protein and lipoprotein associatedphospholipase A2, h) at least one test for genetic testing, said atleast one test for genetic testing being for at least one test for eachof the following: apolipoprotein E genotype, and factor V Leidengenotype, and i) at least one test for secondary causes of highcholesterol, said at least one test for secondary causes of highcholesterol being for at least one test for each of the following:creatinine, blood urea nitrogen, creatine kinase, liver transaminases,alkaline phosphase, thyroid stimulating hormone, and uric acid.